This research program is concerned with elucidation of the basic mechanisms of protective immunity to intracellular bacterial pathogens, including Francisella tularensis, Mycobacterium tuberculosis, and Listeria monocytogenes. A great deal is known about the functional capacities, recognition mechanisms, and signal transduction strategies of inflammatory cells and lymphocytes; relatively less is known about their in vivo contribution to effecting successful host response to infection. A better understanding of the nature of protective immunity is essential to the rational design of new or improved vaccines, and prediction of useful correlates of protection. Thus we are characterizing the pathology of infection, the cell types involved, their cell-cell interactions and products, the specificity of the responses, the recognition receptors used, and the nature of the cellular responses provoked in the context of in vivo primary and secondary intracellular bacterial infection. Animal models that conveniently allow concurrent study of sublethal infection, lethal infection, and immune memory are therefore attractive. Murine infection with Francisella tularensis Live Vaccine Strain (LVS), a gram negative facultative intracellular bacterium that replicates in macrophages and that is of interest as a possible bioterrorism agent, has these features. Protective immune responses to F. tularensis appear quite similar to those of M. tuberculosis and L. monocytogenes. Thus each can be studied as representative of this class of pathogens, with a view toward identifying common or distinct patters of immune responses to intracellular bacteria. To date, study of murine infection with LVS indicates that resolution of primary infection, as well as successful vaccination, is dependent on engagement of innate immunity (TNF alpha and interferon gamma produced by macrophages and natural killer cells), followed by development of either specific CD4+ or CD8+ alpha/beta+ T cells. Studies this year demonstrated that secondary protective immunity to LVS is also clearly dependent on B cells, in that optimal protection against lethal challenge cannot be demonstrated in B cell depleted mice or B cell knockout mice. This defect could be readily reconstituted by transfer of B cells, but not by transfer of immune serum, suggesting that B cells themselves contribute to optimal protection in a non-antibody dependent manner. To determine the specific effector mechanism(s) contributed by B cells, as well as to study the effector mechanism(s) used by T cells (or other cell types) to kill bacteria and/or infected macrophages, we developed an in vitro culture system that replicates many of the features of in vivo infection. Bone marrow macrophages infected with LVS supported exponential growth of bacteria; addition of spleen cells from LVS-immune mice controlled bacterial growth over a four day period. Current studies are dissecting LVS-immune spleen cell populations to determine the exact cell type(s) responsible for control of bacterial growth, the cell surface molecules involved, the means of bacterial killing, and the soluble products involved. In contrast to studies of parenteral infection with LVS, B cell knockout mice given an aerosol infection of a clinical isolate of M. tuberculosis developed less pathology in the lung, and exhibited delayed dissemination of bacteria to the spleen and liver, compared to wild type mice. These delays could again be reconstituted by transfer of B cells, but not specific antibodies, to M. tuberculosis infected B cells knockout mice. The different outcomes in LVS versus M. tuberculosis infection of B cell knockout mice may be due to differences in route of infection and/or trafficking of cells to different sites. Finally, several lines of evidence indicate B cells (but not antibodies) are also important in innate immunity to LVS and Listeria: very strong nonspecific protective immunity to both develops in both normal and T cell knockout mice, but not scid or B cell knockout mice, within 2-3 days after sublethal infection. This early protective immunity, which is clearly nonspecific and a function of innate immunity, can be stimulated by bacterial DNA containing unmethylated CpG motifs. Studies using the in vitro system with DNA-primed spleen cells added to LVS-infected bone marrow macrophages indicate that control of bacterial growth is effected through production of a soluble factor(s), rather than through cell-cell contact. Current studies are dissecting DNA-immune spleen cell populations to determine the exact cell type(s) responsible for control of bacterial growth, the cell surface molecules involved, the means of bacterial killing, and the soluble products involved. Understanding such immunobiological properties of bacterial DNA will be important in evaluation of DNA vaccines, the use of bacterial DNA as an adjuvant, and therapeutic applications of bacterial DNA.